1 Setup

This section and the next are relevant for reproducibility purposes. For results, please skip to section 3 (Quality Control)

  • Libraries
suppressPackageStartupMessages({
  library(data.table)
  library(DESeq2)
  library(emoji)
  library(gplots)
  library(gtools)
  library(here)
  library(hyperSpec)
  library(limma)
  library(magrittr)
  library(parallel)
  library(patchwork)
  library(PCAtools)
  library(pheatmap)
  library(plotly)
  library(pvclust)
  library(RColorBrewer)
  library(tidyverse)
  library(tximport)
  library(vsn)
})
  • Helper functions
source("/mnt/picea/home/fai/git/UPSCb-common/src/R/featureSelection.R")
  • Graphics
hpal <- colorRampPalette(c("blue","white","red"))(100)
pal <- brewer.pal(9,"Blues")

2 Data

  • Sample information
samples <- read_csv(here("doc/spruce.csv"),
                    col_types=cols(.default=col_factor())) %>%
  separate_wider_delim(SampleDescription,delim = " - ",names = c("EcotypeLatitude","LightCondition","Replicate")) %>%
  mutate(EcotypeLatitude = gsub(" (latitude)","",gsub("Spruce ecotype ","S",EcotypeLatitude),fixed = T),
         LightCondition = gsub("light condition ","",LightCondition),
         Replicate = gsub("replicate ","",Replicate))
  • tx2gene translation table
tx2gene <- suppressMessages(read_delim(here("analysis/spruce/nfcoreResult/salmon/tx2gene.tsv"),delim="\t",
                                 col_names=c("TXID","GENE"))) %>%
  select(-X3)
  • Raw data
filelist <- data.frame(Filenames = list.files(here("analysis/spruce/nfcoreResult/salmon"), 
                          recursive = TRUE, 
                          pattern = "quant.sf",
                          full.names = TRUE)) %>%
  mutate(SampleID = basename(dirname(Filenames)))
  • Sanity check to ensure that the data is sorted according to the sample info
# stopifnot(all(match(sub("_CHANGE-ME.*","",basename(dirname(filelist))),
#                     samples$SampleID) == 1:nrow(samples)))
  • add filelist to samples as a new column
samples %<>% left_join(filelist, by = "SampleID")
  • export full rank samples
write_tsv(samples,here("doc/samples_full_rank_spruce.txt"))

Read the expression at the gene level

txi <- suppressMessages(tximport(files = samples$Filenames,
                                 type = "salmon",
                                 tx2gene=tx2gene))
counts <- txi$counts
colnames(counts) <- samples$SampleID

 

3 Quality Control

  • “Not expressed” genes
sel <- rowSums(counts) == 0
sprintf("%s%% percent (%s) of %s genes are not expressed",
        round(sum(sel) * 100/ nrow(counts),digits=1),
        sum(sel),
        nrow(counts))
## [1] "9.5% percent (5136) of 54130 genes are not expressed"

3.1 Sequencing depth

  • Let us take a look at the sequencing depth
dat <- tibble(x=colnames(counts),y=colSums(counts)) %>% 
  bind_cols(samples)

ggplot(dat,aes(x,y,fill=EcotypeLatitude)) + 
  geom_col() + 
  scale_y_continuous(name="reads") +
  facet_grid(~ factor(LightCondition), scales = "free") +
  theme_bw() + 
  theme(axis.text.x=element_text(angle=90,size=8),
        axis.title.x=element_blank())

👉 The raw sequencing depth of replicate 4-6 are significantly lower than replicate 1-3.

3.2 per-gene mean expression

i.e. the mean raw count of every gene across samples is calculated and displayed on a log10 scale.

ggplot(data.frame(value=log10(rowMeans(counts))),aes(x=value)) + 
  geom_density(na.rm = TRUE) +
  ggtitle("gene mean raw counts distribution") +
  scale_x_continuous(name="mean raw counts (log10)") + 
  theme_bw()

👉 The cumulative gene coverage is as expected

3.3 Per-sample expression

dat <- as.data.frame(log10(counts)) %>% 
  utils::stack() %>% 
  mutate(LightCondition=samples$LightCondition[match(ind,samples$SampleID)],
         Ecotype=samples$EcotypeLatitude[match(ind,samples$SampleID)],
         Replicate=samples$Replicate[match(ind,samples$SampleID)])

ggplot(dat,aes(x=values,group=ind,col=LightCondition)) + 
  geom_density(na.rm = TRUE) + 
  ggtitle("sample raw counts distribution") +
  scale_x_continuous(name="per gene raw counts (log10)") + 
  theme_bw()

ggplot(dat,aes(x=values,group=ind,col=Ecotype)) + 
  geom_density(na.rm = TRUE) + 
  ggtitle("sample raw counts distribution") +
  scale_x_continuous(name="per gene raw counts (log10)") + 
  theme_bw()

ggplot(dat,aes(x=values,group=ind,col=Replicate)) + 
  geom_density(na.rm = TRUE) + 
  ggtitle("sample raw counts distribution") +
  scale_x_continuous(name="per gene raw counts (log10)") + 
  theme_bw()

👉 Replicate 4-6 has different sequencing depth characteristics from replicate 1-3.

  • Export raw expression data
# dir.create(here("data/analysis/salmon"),showWarnings=FALSE,recursive=TRUE)
write.csv(counts,file=here("analysis/spruce/raw-unormalised-gene-expression_data.csv"))

 

4 Data normalisation

4.1 Preparation

For visualization, the data is submitted to a variance stabilization transformation using DESeq2. The dispersion is estimated independently of the sample tissue and replicate.

dds <- DESeqDataSetFromTximport(
  txi=txi,
  colData = samples,
  design = ~ LightCondition)
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in design formula are characters, converting to factors
## using counts and average transcript lengths from tximport
colnames(dds) <- samples$SampleID

save(dds,file=here("analysis/spruce/dds.rda"))

4.2 size factors

(i.e. the sequencing library size effect)

dds <- estimateSizeFactors(dds) %>% 
  suppressMessages()

boxplot(normalizationFactors(dds),
        main="Sequencing libraries size factor",
        las=2,log="y")
abline(h=1, col = "Red", lty = 3)

and without outliers:

boxplot(normalizationFactors(dds),
        main="Sequencing libraries size factor",
        las=2,log="y", outline=FALSE)
abline(h=1, col = "Red", lty = 3)

👉 The libraries’ size factors of replicate 4-6 are significantly lower than replicate 1-3.

Assess whether there might be a difference in library size linked to a given metadata

boxplot(split(t(normalizationFactors(dds)),dds$Replicate),las=2,
        main="Sequencing libraries size factor by Replicate",
        outline=FALSE)

boxplot(split(t(normalizationFactors(dds[,dds$Replicate %in% 1:3])),dds[,dds$Replicate %in% 1:3]$EcotypeLatitude),las=2,
        main="Sequencing libraries size factor by Ecotype in replicate 1-3",
        outline=FALSE)

boxplot(split(t(normalizationFactors(dds[,dds$Replicate %in% 1:3])),dds[,dds$Replicate %in% 1:3]$LightCondition),las=2,
        main="Sequencing libraries size factor by Light condition in replicate 1-3",
        outline=FALSE)

boxplot(split(t(normalizationFactors(dds[,dds$Replicate %in% 4:6])),dds[,dds$Replicate %in% 4:6]$EcotypeLatitude),las=2,
        main="Sequencing libraries size factor by Ecotype in replicate 4-6",
        outline=FALSE)

boxplot(split(t(normalizationFactors(dds[,dds$Replicate %in% 4:6])),dds[,dds$Replicate %in% 4:6]$LightCondition),las=2,
        main="Sequencing libraries size factor by Light condition in replicate 4-6",
        outline=FALSE)

👉 The scaling factor distribution is dependent of replicate, but not dependent of other variables.

plot(colMeans(normalizationFactors(dds)),
     log10(colSums(counts(dds))),ylab="log10 raw depth",
     xlab="average scaling factor",
     col=rainbow(n=6)[as.integer(dds$Replicate)],pch=19)
legend("bottomright",fill=rainbow(n=6),
       legend=unique(dds$Replicate),cex=0.6)

👉 The scaling factor appear linearly proportional to the sequencing depth in all replicates.

4.3 Variance Stabilising Transformation

vsd <- varianceStabilizingTransformation(dds, blind=TRUE)
vst <- assay(vsd)
vst <- vst - min(vst)

4.4 Validation

let’s look at standard deviations before and after VST normalization. This plot is to see whether there is a dependency of SD on the mean.

Before:

meanSdPlot(log1p(counts(dds))[rowSums(counts(dds))>0,])

After:

meanSdPlot(vst[rowSums(vst)>0,])

After VST normalization, the red line is almost horizontal which indicates no dependency of variance on mean (homoscedastic).

👉 We can conclude that the variance stabilization worked adequately

 

4.5 QC on the normalised data

4.5.1 PCA

pc <- prcomp(t(vst))
percent <- round(summary(pc)$importance[2,]*100)

Using PCAtools

p <- pca(vst,colData(dds))

4.5.2 Scree plot

We define the number of variable of the model:

nvar <- 1

An the number of possible combinations

nlevel <- 3

We devise the optimal number of components using two methods

horn <- suppressWarnings(parallelPCA(vst))
elbow <- findElbowPoint(p$variance)

We plot the percentage explained by different components and try to empirically assess whether the observed number of components would be in agreement with our model’s assumptions.

  • the red line represents number of variables in the model
  • the orange line represents number of variable combinations.
  • the black dotted, annotate lines represent the optimal number of components reported by the horn and elbow methods.
ggplot(tibble(x=1:length(percent),y=cumsum(percent),p=percent),aes(x=x,y=y)) +
  geom_line() + geom_col(aes(x,p)) + scale_y_continuous("variance explained (%)",limits=c(0,100)) +
  scale_x_continuous("Principal component",breaks=1:length(percent),minor_breaks=NULL) + 
  geom_vline(xintercept=nvar,colour="red",linetype="dashed",linewidth=0.5) + 
  geom_hline(yintercept=cumsum(percent)[nvar],colour="red",linetype="dashed",linewidth=0.5) +
  geom_vline(xintercept=nlevel,colour="orange",linetype="dashed",linewidth=0.5) + 
  geom_hline(yintercept=cumsum(percent)[nlevel],colour="orange",linetype="dashed",linewidth=0.5) +
  geom_vline(xintercept=c(horn$n,elbow),colour="black",linetype="dotted",linewidth=0.5) +
  geom_label(aes(x = horn$n + 1, y = cumsum(percent)[horn$n],label = 'Horn', vjust = 1)) +
  geom_label(aes(x = elbow + 1, y = cumsum(percent)[elbow],label = 'Elbow', vjust = 1))
## Warning in geom_label(aes(x = horn$n + 1, y = cumsum(percent)[horn$n], label = "Horn", : All aesthetics have length 1, but the data has 18 rows.
## ℹ Please consider using `annotate()` or provide this layer with data containing a single row.
## Warning in geom_label(aes(x = elbow + 1, y = cumsum(percent)[elbow], label = "Elbow", : All aesthetics have length 1, but the data has 18 rows.
## ℹ Please consider using `annotate()` or provide this layer with data containing a single row.

👉 The first component explains 40% of the data variance. Both metrics, Horn and Elbow suggest that one or two components are those that are informative. Indeed the slope of the curve is fairly linear past PC3 and that would indicate that the remaining PCs only capture sample specific noise. While this is only empirical, the scree plot support having only few variables of importance in the dataset.

4.5.3 PCA plot

pc.dat <- bind_cols(PC1=pc$x[,1],
                    PC2=pc$x[,2],
                    PC3=pc$x[,3],
                    as.data.frame(colData(dds)))
  • PC1 vs PC2
p1 <- ggplot(pc.dat,aes(x=PC1,y=PC2,col=LightCondition,shape=EcotypeLatitude,text=SampleID)) + 
  geom_point(size=2) + 
  ggtitle("Principal Component Analysis",subtitle="variance stabilized counts")

ggplotly(p1) %>% 
  layout(xaxis=list(title=paste("PC1 (",percent[1],"%)",sep="")),
         yaxis=list(title=paste("PC2 (",percent[2],"%)",sep=""))) %>% suppressWarnings()

The same as a biplot

biplot(p,
       colby = 'LightCondition',
       colLegendTitle = 'LightCondition',
       encircle = TRUE,
       encircleFill = TRUE,
       legendPosition = 'top', 
       legendLabSize = 16, legendIconSize = 8.0)
## Registered S3 methods overwritten by 'ggalt':
##   method                  from   
##   grid.draw.absoluteGrob  ggplot2
##   grobHeight.absoluteGrob ggplot2
##   grobWidth.absoluteGrob  ggplot2
##   grobX.absoluteGrob      ggplot2
##   grobY.absoluteGrob      ggplot2

biplot(p,
       colby = 'EcotypeLatitude',
       colLegendTitle = 'EcotypeLatitude',
       encircle = TRUE,
       encircleFill = TRUE,
       legendPosition = 'top', 
       legendLabSize = 16, legendIconSize = 8.0)

👉 ****PC1 (41%) explains light condition. PC2 (11%) explain the ecotypes.****

  • PC1 vs PC3
p2 <- ggplot(pc.dat,aes(x=PC1,y=PC3,col=LightCondition,shape=EcotypeLatitude,text=SampleID)) + 
  geom_point(size=2) + 
  ggtitle("Principal Component Analysis",subtitle="variance stabilized counts")

ggplotly(p2) %>% 
  layout(xaxis=list(title=paste("PC1 (",percent[1],"%)",sep="")),
         yaxis=list(title=paste("PC3 (",percent[3],"%)",sep=""))) %>% suppressWarnings()

The same as a biplot

biplot(p,x = 'PC1', y = 'PC3',
       colby = 'LightCondition',
       colLegendTitle = 'LightCondition',
       encircle = TRUE,
       encircleFill = TRUE,
       legendPosition = 'top', 
       legendLabSize = 16, legendIconSize = 8.0)

biplot(p,x = 'PC1', y = 'PC3',
       colby = 'EcotypeLatitude',
       colLegendTitle = 'EcotypeLatitude',
       encircle = TRUE,
       encircleFill = TRUE,
       legendPosition = 'top', 
       legendLabSize = 16, legendIconSize = 8.0)

👉 PC1 (41%) explains light condition. PC2 (11%) explain the ecotypes. PC3 (7.6%) seems to explain the difference of A and B.

4.5.4 Pairs plot

This allows for looking at more dimensions, five by default

suppressMessages(pairsplot(p,colby='LightCondition',shape='EcotypeLatitude'))

4.5.5 Loadings

Loadings, i.e. the individual effect of every gene in a component can be studied. Here the most important ones are visualized throughout the different PCs

plotloadings(p,
             rangeRetain = 0.01,
             labSize = 4.0,
             title = 'Loadings plot',
             subtitle = 'PC1 to PC5',
             caption = 'Top 1% variables',
             drawConnectors = TRUE)
## -- variables retained:
## PA_chr12_G001223, PA_chr09_G004291, PA_sUP004_G000022, PA_chr07_G005145, PA_chr07_G004145, PA_chr07_G002686, PA_chr02_G005328, PA_chr11_G003044, PA_chr02_G005240
## Warning: ggrepel: 9 unlabeled data points (too many overlaps). Consider increasing max.overlaps

4.5.6 Correlation

This is a plot showing the correlation between the PC and the model variables. Note that while this might be relevant for a linear variable, it is less so for categorical variables. Sorting categorical variables in a linear order according to the PCs above might help.

{r beffect, echo=FALSE, eval=FALSE # You could do as follows to reorder a categorial variable p$metadata$Beffect <- ifelse(as.integer(dds$Batch)==1,3,as.integer(dds$Batch)-1)

Plotting only the relevant variables.

suppressWarnings(eigencorplot(p,metavars=c('LightCondition','EcotypeLatitude','Replicate')))

👉 PC1 and PC3 explain light conditions. PC2 explains ecotypes. PC1 is also affected by replicate.

4.5.7 Samples Distance

sampleDists <- dist(t(assay(vsd)))
sampleDistMatrix <- as.matrix(sampleDists)
rownames(sampleDistMatrix) <- colnames(sampleDistMatrix) <- dds$SampleID
hm <- pheatmap(sampleDistMatrix,
         clustering_distance_rows=sampleDists,
         clustering_distance_cols=sampleDists,
         col=pal)
hm

👉 Samples cluster according to whether the light condition is D or A/B. Within the A/B clade, the samples cluster according to ecotype.

4.6 Sequencing depth

The figures show the number of genes expressed per condition at different expression cutoffs. The scale on the lower plot is the same as on the upper. The first plot is a heatmap showing the number of genes above a given cutoff. The second plot shows it as a ratio of the number of genes expressed for (a) given variable(s) divided by the average number of genes expressed at that cutoff across all variable(s). The latter plot is of course biased at higher cutoff as the number of genes becomes smaller and smaller. The point of these two plots is to assert whether the number of genes expressed varies between conditions, as this would break some assumptions for normalisation and differential expression.

conds <- factor(paste(dds$LightCondition,dds$EcotypeLatitude))
dev.null <- rangeSamplesSummary(counts=vst,
                                conditions=conds,
                                nrep=3)

Plotting the number of genes that are expressed (at any level)

(nrow(vst) - colSums(vst==0)) %>% as.data.frame() %>% 
  rownames_to_column("SampleID") %>% 
  rename("ExpressedGenes"=".") %>% 
  left_join(samples,by="SampleID") %>% 
  ggplot(aes(x=LightCondition, y=ExpressedGenes, fill=EcotypeLatitude)) + 
  geom_dotplot(binaxis = "y", stackdir = "center")
## Bin width defaults to 1/30 of the range of the data. Pick better value with `binwidth`.

👉 The number of genes expressed is affected by ecotypes but not light condition.

4.7 Heatmap

Here we want to visualise all the informative genes as a heatmap. We first filter the genes to remove those below the selected noise/signal cutoff. The method employed here is naive, and relies on observing a sharp decrease in the number of genes within the first few low level of expression. Using an independent filtering method, such as implemented in DESeq2 would be more accurate, but for the purpose of QA validation, a naive approach is sufficient. Note that a sweet spot for computation is around 20 thousand genes, as building the hierarchical clustering for the heatmap scales non-linearly.

sels <- rangeFeatureSelect(counts=vst,
                           conditions=conds,
                           nrep=3) %>% 
  suppressWarnings()

vst.cutoff <- 1

👉 Here a cutoff of 1 is applied

nn <- nrow(vst[sels[[vst.cutoff+1]],])
tn <- nrow(vst)
pn <- round(nn * 100/ tn, digits=1)

⚠️ 57.1% (30898) of total 54130 genes are plotted below:

mat <- t(scale(t(vst[sels[[vst.cutoff+1]],])))
hm <- pheatmap(mat,
               color = hpal,
               border_color = NA,
               clustering_distance_rows = "correlation",
               clustering_method = "ward.D2",
               show_rownames = FALSE,
               #labels_col = conds,
               angle_col = 90,
               legend = FALSE)
hm

👉 By selecting only informative genes, We still see the separation of D, and ecotype-clustering within A/B clade. We can see the difference in ecotypes within D clade also.

4.8 Clustering of samples

Below we assess the previous dendrogram’s reproducibility and plot the clusters with au and bp where:

  • au (Approximately Unbiased): computed by multiscale bootstrap resampling 👈 a better approximation
  • bp (Bootstrap Probability): computed by normal bootstrap resampling

⚠️Clusters with AU larger than 95% are highlighted by rectangles, which are strongly supported by data

hm.pvclust <- pvclust(data = t(scale(t(vst[sels[[vst.cutoff+1]],]))),
                       method.hclust = "ward.D2", 
                       nboot = 100, parallel = TRUE)
## Creating a temporary cluster...done:
## socket cluster with 15 nodes on host 'localhost'
## Multiscale bootstrap... Done.
plot(hm.pvclust)#, labels = conds)
pvrect(hm.pvclust)

👉 D is separated from A/B. Within D clade and in A/B clade, samples cluster based on ecotypes.

bootstrapping results as a table 
print(hm.pvclust, digits=3)
## 
## Cluster method: ward.D2
## Distance      : correlation
## 
## Estimates on edges:
## 
##       si    au    bp se.si se.au se.bp      v      c  pchi
## 1  1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 2  1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 3  1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 4  1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 5  1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 6  0.945 0.970 0.982 0.061 0.039 0.005 -1.989 -0.109 0.993
## 7  1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 8  1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 9  1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 10 0.629 0.782 0.889 0.120 0.091 0.011 -1.000 -0.220 0.211
## 11 1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 12 1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 13 1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 14 1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 15 1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 16 1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000
## 17 1.000 1.000 1.000 0.000 0.000 0.000  0.000  0.000 0.000

5 Session Info

Session Info 
sessionInfo()
## R version 4.4.2 (2024-10-31)
## Platform: x86_64-pc-linux-gnu
## Running under: Ubuntu 22.04.5 LTS
## 
## Matrix products: default
## BLAS/LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.20.so;  LAPACK version 3.10.0
## 
## locale:
##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C               LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8    LC_PAPER=en_US.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C             LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       
## 
## time zone: Europe/Stockholm
## tzcode source: system (glibc)
## 
## attached base packages:
##  [1] parallel  grid      stats4    stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
##  [1] vsn_3.74.0                  tximport_1.34.0             RColorBrewer_1.1-3          pvclust_2.2-0              
##  [5] plotly_4.10.4               pheatmap_1.0.12             PCAtools_2.18.0             ggrepel_0.9.6              
##  [9] patchwork_1.3.0             magrittr_2.0.3              limma_3.62.1                hyperSpec_0.100.2          
## [13] xml2_1.3.6                  lattice_0.22-6              here_1.0.1                  gtools_3.9.5               
## [17] gplots_3.2.0                emoji_16.0.0                DESeq2_1.46.0               SummarizedExperiment_1.36.0
## [21] Biobase_2.66.0              MatrixGenerics_1.18.0       matrixStats_1.4.1           GenomicRanges_1.58.0       
## [25] GenomeInfoDb_1.42.0         IRanges_2.40.0              S4Vectors_0.44.0            BiocGenerics_0.52.0        
## [29] data.table_1.16.2           lubridate_1.9.3             forcats_1.0.0               stringr_1.5.1              
## [33] dplyr_1.1.4                 purrr_1.0.2                 readr_2.1.5                 tidyr_1.3.1                
## [37] tibble_3.2.1                ggplot2_3.5.1               tidyverse_2.0.0            
## 
## loaded via a namespace (and not attached):
##  [1] rstudioapi_0.17.1         jsonlite_1.8.9            farver_2.1.2              rmarkdown_2.29           
##  [5] zlibbioc_1.52.0           vctrs_0.6.5               DelayedMatrixStats_1.28.0 htmltools_0.5.8.1        
##  [9] S4Arrays_1.6.0            SparseArray_1.6.0         proj4_1.0-14              sass_0.4.9               
## [13] KernSmooth_2.23-24        bslib_0.8.0               htmlwidgets_1.6.4         plyr_1.8.9               
## [17] testthat_3.2.1.1          cachem_1.1.0              ggalt_0.4.0               lifecycle_1.0.4          
## [21] pkgconfig_2.0.3           rsvd_1.0.5                Matrix_1.7-1              R6_2.5.1                 
## [25] fastmap_1.2.0             GenomeInfoDbData_1.2.13   digest_0.6.37             colorspace_2.1-1         
## [29] rprojroot_2.0.4           dqrng_0.4.1               irlba_2.3.5.1             crosstalk_1.2.1          
## [33] beachmat_2.22.0           labeling_0.4.3            fansi_1.0.6               timechange_0.3.0         
## [37] httr_1.4.7                abind_1.4-8               compiler_4.4.0            bit64_4.5.2              
## [41] withr_3.0.2               BiocParallel_1.40.0       hexbin_1.28.4             Rttf2pt1_1.3.12          
## [45] maps_3.4.2                MASS_7.3-61               DelayedArray_0.32.0       caTools_1.18.3           
## [49] tools_4.4.0               extrafontdb_1.0           glue_1.8.0                reshape2_1.4.4           
## [53] generics_0.1.3            gtable_0.3.6              tzdb_0.4.0                preprocessCore_1.68.0    
## [57] hms_1.1.3                 BiocSingular_1.22.0       ScaledMatrix_1.14.0       utf8_1.2.4               
## [61] XVector_0.46.0            pillar_1.9.0              vroom_1.6.5               bit_4.5.0                
## [65] deldir_2.0-4              tidyselect_1.2.1          locfit_1.5-9.10           knitr_1.49               
## [69] xfun_0.49                 statmod_1.5.0             brio_1.1.5                stringi_1.8.4            
## [73] UCSC.utils_1.2.0          lazyeval_0.2.2            yaml_2.3.10               evaluate_1.0.1           
## [77] codetools_0.2-20          interp_1.1-6              extrafont_0.19            BiocManager_1.30.25      
## [81] cli_3.6.3                 affyio_1.76.0             ash_1.0-15                munsell_0.5.1            
## [85] jquerylib_0.1.4           Rcpp_1.0.13-1             png_0.1-8                 latticeExtra_0.6-30      
## [89] jpeg_0.1-10               sparseMatrixStats_1.18.0  bitops_1.0-9              viridisLite_0.4.2        
## [93] scales_1.3.0              affy_1.84.0               crayon_1.5.3              rlang_1.1.4              
## [97] cowplot_1.1.3

 

 

drawing

Created by Fai Theerarat Kochakarn

theerarat dot kochakarn at umu dot se